Nicotine is a powerfully addictive drug and, as the primary pharmacological agent in tobacco products, is the basis of a serious world-wide health problem. Part of the effort against this problem is the attempt to understand the basis of the addictive process. In order to do that we need to know more about the molecular sites of action of nicotine. We have focused on what appears to be the major nicotinic subtype in brain - known as the alpha4 nAChR. In order to study the mechanism of assembly and subsequent regulation of this receptor, we have developed a transient transfection system that allows different combinations of subunits to be expressed in mammalian cells (COS). The system is rapid, reproducible and very flexible. We have found that the alpha4 subunit acquires high affinity nicotine binding when expressed with the beta2 subunit but lower affinity when expressed with the beta4 subunit. However, the alpha4beta4 combination gives 10-15 fold more ligand binding than alpha4beta2. Expression of alpha4 alone does not result in any ligand binding. If alpha4 is co-expressed with beta2 and beta4, the nicotine binding assay indicates the presence of only a low affinity receptor. The addition of the beta3 subunit to a combination of alpha4, beta2 and beta4 results in a dramatic up-regulation of the high affinity site such that the COS cells now express approximately equal amounts of high and low affinity sites similar in kD to the high affinity alpha4beta2 and the low affinity alpha4beta4 site. If either beta4 or beta2 is omitted from the transfection, the binding affinity and receptor number are indistinguishable from transfection with just two subunits. Thus, in order to create the composite receptor with two equally abundant sites of differing affinity, cells must be transfected with alpha4beta2beta3beta4. We plan to use this system to explore three aspects of nicotine action by (1) characterizing the pharmacology of the alpha4 nAChR more fully, (2) studying subunit assembly requirements, (3) analyzing the basis of the difference in ligand affinity between alpha4beta2 and alpha4beta4, and (4) studying the role of phosphorylation in alpha4 nAChR regulation.